New England Biolabs, M0467S, Salt-T4® DNA Ligase

Looking for additional T4 DNA Ligase formulations and variants? Related Categories DNA Ligases Applications BioBrick, ®, Assembly,, Cloning Ligation,, NEBridge® Golden Gate Assembly Specification Unit Definition One unit is defined as the amount of enzyme required to give 50% ligation of 6 µg of Lambda-HindIII DNA in 30 minutes at 25°C in a total reaction volume of 20 µ in 1X T4 DNA Ligase Reaction Buffer supplemented with 100 mM NaCl. Reaction Conditions 1X T4 DNA Ligase Reaction Buffer Incubate at 25°C T4 DNA Ligase Reaction Buffer 50 mM Tris-HCl 10 mM MgCl2 1 mM ATP 10 mM DTT (pH 7.5 @ 25°C) Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 10 minutes FAQ Q: What are some causes of ligation reaction failure that can lead to transformation failure? A: Ligation failed because there was no ATP or Mg2+. Use the supplied buffer or add ATP to a compatible buffer. ATP in buffers older than one year may have degraded enough to cause problems. When supplementing with ATP, be sure to use riboATP as deoxyriboATP will not work. Ligation failed due to EDTA in the reaction. Clean up the DNA (we recommend Monarch® nucleic acid purification kits). CIP, BAP, Antarctic Phosphatase or SAP are not completely inactivated following dephosphorylation step. Follow the recommended procedure to remove the phosphatase. Concentration was too high resulting in ligation only producing linear DNA. Keep the total DNA concentration between 1-10 μg/ml. The insert and the plasmid do not have phosphates. Note: primers may not have phosphates, preventing blunt-end ligation with vectors that have been treated with CIP. Order primers with phosphates or phosphorylate the primers with T4 Polynucleotide Kinase prior to use. Too much ligation mixture was added to the cells. Add between 1-5 μl to 50 μl competent cells. The insert was large and the conditions didn't allow circularization. Reduce the insert concentration. The ligase was inactive. Test on Lambda HindIII DNA or other convenient substrate. The ligation mix contained PEG and was incubated overnight. Extended ligation with PEG causes a drop off in transformation efficiency. This could be due to the gradual production of large linear pieces of DNA that can inhibit transformation. StickTogether™ DNA Ligase Buffer contains PEG. The ligation mix was not purified prior to electroporation. The buffer must be removed or a spark will be generated by the salt. Dialyze the sample or use a spin column to purify (we recommend Monarch nucleic acid purification kits). The PEG in StickTogether™ DNA Ligase Buffer prevents sparking but it also prevents electroporation. PEG must be removed using a spin column. Q: When should Salt-T4 DNA Ligase be the enzyme of choice? A: Whereas T4 DNA Ligase is sensitive to the presence of salt in the reaction, Salt-T4 DNA Ligase can tolerate salt concentrations up to 500 mM for the ligation of cohesive ends, or up to 150 mM for the ligation of blunt ends. Therefore, Salt-T4 DNA Ligase should be the enzyme of choice if the ligation is to be performed in a salt-containing buffer (e.g., NEBuffer 3.1) or if salt carryover (from DNA preparations) is a concern. Please note in the presence of PEG (1X StickTogether™ DNA Ligase Buffer), DNA in a high salt solution will precipitate, inhibiting ligation. If you want to ligate DNA in the presence of salt over 100 mM, we recommend the use of 1X T4 DNA Ligase Buffer. Q: Which buffer should I use with Salt-T4 DNA Ligase? A: If trying to ligate cohesive ends or blunt ends in the presence of >100 mM salt, we recommend using Salt-T4 DNA Ligase in 1X T4 DNA Ligase Buffer. If trying to ligate blunt ends or single-base overhangs, Salt-T4 DNA Ligase should be used in StickTogether™ DNA Ligase Buffer. We do not recommend supplementing the StickTogether™ DNA Ligase Buffer with > 100 mM salt, as PEG in the StickTogether™ DNA Ligase Buffer and DNA will precipitate, inhibiting ligation. If trying to ligate DNA in the presence of >100 mM salt, we recommend using 1X T4 DNA Ligase Buffer. Do not heat kill the reaction, as heat treating the PEG in the StickTogether™ DNA Ligase Buffer will inhibit transformation. Q: What is the activity of Salt-T4 DNA Ligase in other buffers? A: Salt-T4 DNA Ligase is 100% active in T4 DNA Ligase Buffer supplemented with 100 mM NaCl, NEBuffer r3.1 and HiFi Taq DNA Ligase Buffer (when supplemented with ATP). Salt-T4 DNA Ligase has reduced activity in other NEB restriction enzyme and ligase buffers, however, activity in these buffers can be restored when supplemented with 100 mM NaCl. Buffer % Activity1 T4 DNA Ligase Buffer supplemented with 100 mM NaCl 100% T4 DNA Ligase Buffer 25% NEBuffer r1.12 25% NEBuffer r1.12 supplemented with 100 mM NaCl 100% NEBuffer r2.12 50% NEBuffer r2.12 supplemented with 100 mM NaCl 100% NEBuffer r3.12 100% rCutSmart® Buffer2 25% rCutSmart Buffer2 supplemented with 100 mM NaCl 100% Taq DNA Ligase Buffer2 25% HiFi Taq DNA Ligase Buffer2 100% 1Activity relative to activity on a cohesive end substrate in T4 DNA Ligase Buffer supplemented with 100 mM NaCl (25°C for 10 minutes). 2 Supplemented with 1 mM ATP. Please be sure to use riboATP (NEB #P0756) as deoxyriboATP will not work. To see its % functional activity in CutSmart, and that of other DNA Modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart. Q: Can this ligase be heat inactivated? A: Yes, just like T4 DNA Ligase, this ligase can be heat inactivated for 10 minutes at 65°C. Do not heat inactivate if there is PEG in the reaction buffer (StickTogether™ DNA Ligase Buffer), as transformation will be inhibited. Q: What is the difference between the unit definitions of T4 DNA Ligase (NEB #M0202) and Salt-T4 DNA Ligase (NEB #M0467)? A: Both T4 DNA Ligase and Salt-T4 DNA Ligase unit definitions are defined as 50% ligation of 0.12 μM HindIII fragments, however, the incubation time/temperature, buffer conditions as well as the method of detection of ligation products differs. The T4 DNA Ligase unit definition is determined after 30 minutes at 16°C, while the Salt-T4 DNA Ligase unit is determined after 10 minutes at 25°C. The T4 DNA Salt-T4 DNA Ligase and T4 DNA Ligase have the same specific activity (1 unit of Salt-T4 DNA Ligase in 1X T4 DNA Ligase Buffer + 100 mM NaCl=1 unit of T4 DNA Ligase in 1X T4 DNA Ligase Buffer). Q: How much DNA should be used in a ligation using this ligase? A: The unit definition uses 6 μg HindIII fragment. This high DNA concentration can be used for linker ligation. To promote circle formation, useful in transformation, a lower total DNA concentration should be used. The overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Vector:Insert molar ratios between 1:1 and 1:10 are recommended (1:3 is typical). If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios. Q: Does Salt-T4 DNA Ligase have a higher tolerance to salt than other ligases? A: Yes. While T3 DNA Ligase can tolerate salt concentrations up to 250-300 mM in the reaction, Salt-T4 DNA Ligase can tolerate salt concentrations up to 500 mM in the reaction for cohesive end ligation and up to 150 mM salt concentrations for blunt-end ligation. Please note in the presence of PEG (e.g., StickTogether™ DNA Ligase Buffer), DNA in a high salt solution will precipitate, inhibiting ligation. If you want to ligate DNA in the presence of >100 mM salt, we recommend 1X T4 DNA Ligase Buffer. Q: Can the ligation reaction produced by Salt-T4 DNA Ligase be directly used to transform electrocompetent cells? A: We recommend purification of the ligation reaction prior to electroporation. The buffer must be removed or a spark will be generated by the salt present. Dialyze the sample or use a spin column to purify (we recommend Monarch nucleic acid purification kits). The PEG in StickTogether™ DNA Ligase Buffer prevents sparking, but it also prevents electroporation and must be removed by a spin column.